1. Alginate Electrodeposition
2. Codeposition of Communicating Cell Populations in Alginate
3. Chitosan Electrodeposition
4. Electrochemical Transduction with a Functionalized Chitosan Film
5. Protein Functionalization Using Enzymatic Assembly
6. Representative Results
Imposed electrical signals can create localized microenvironments (e.g., fields and gradients) near an electrode surface and these stimuli can trigger the self-assembly of polysaccharides such as alginate and chitosan to deposit as a hydrogel film on the electrode surface. Because this sol-gel transition occurs at the electrode surface, the resulting film is electroaddressed, with its geometry matching the electrode pattern (Figures 1B, 3C). Biocompatible films such as alginate and chitosan provide surfaces that can be functionalized with biological components. Using alginate, unique cell populations have been codeposited at separate addresses. Evidence of their electroaddressment is observed upon interaction between the sender and receiver cell population. The molecule autoinducer-2 (AI-2) diffuses from the sender cells and is taken up by the receiver cells, resulting in expression of the dsRed red fluorescent protein (Figure 2A). In Figure 2B, red fluorescence is observed only at the electrode where receivers are addressed.
The amine groups present on chitosan provide it with the pH responsiveness required for electrodeposition as well as a surface suitable for functionalization. We utilized these unique properties by electrochemically conjugating the biosensing enzyme glucose oxidase (GOx) to electrodeposited chitosan films. This enzyme then provides the ability for detection of glucose through an enzymatic reaction (Figure 4A) producing hydrogen peroxide which can then be electrochemically oxidized to produce an output current. In this way, a chemical signal can be transduced to electrical. Figure 4B shows that films in which GOx was electrochemically conjugated produce a strong anodic signal in the presence of glucose as opposed to those films containing no GOx. These results indicate GOx can be assembled onto a deposited chitosan film and will retain catalytic activity. Furthermore, Figure 4C shows a step-increase in anodic current produced in response to increasing glucose concentrations. The standard curve also present in Figure 4C shows that the step-increases proceeded in a near linear fashion dependent on the amount of glucose added. These results show that the enzyme also retains its sensitivity to increasing glucose concentrations upon conjugation to the chitosan film. The lower limit of detection was not studied here as it has been previously characterized for this system in the work of Meyer et. al.
We also have demonstrated the covalent immobilization of an enzyme of interest, engineered to contain a custom penta-tyrosine tag, to chitosan in an enzymatically-controlled manner. Specifically, this process is mediated by the enzyme tyrosinase. As depicted by the scheme in Figure 5A(upper), an enzyme, AI-2 Synthase includes a penta-tyrosine tag. Tyrosinase acts on the tyrosine tag, oxidizing the residues’ phenol groups to O-quinones, which then covalently bind to chitosan’s amines. Evidence of chitosan film functionalization with the AI-2 Synthase by tyrosinase assembly is observed in Figure 5B, where the AI-2 Synthase has been fluorescently labeled blue. Because AI-2 Synthase generates AI-2 from the substrate S-adenosyl homocysteine (SAH) in the same way as the sender cells, its proximity to codeposited receiver cells in the presence of SAH also causes the receiver cells to fluorescently respond by expressing dsRed (Figure 5A(lower)). Red fluorescence of the receiver cells (Figure 5B) again demonstrates interaction between addresses due to the diffusion of AI-2 from one to the other, and further indicates that enzymes immobilized to chitosan retain activity once covalently bound.
Figure 1. Alginate electrodeposition. (A) Mechanism of alginate electrodeposition: As an electrode is anodically biased, water electrolysis occurs at its surface, generating a localized low pH. Calcium carbonate particles react with the surplus of protons, releasing calcium cations as the particles dissolve. In the presence of alginate polymer chains, the ions become chelated in an “eggbox” network, forming a crosslinked hydrogel at the electrode. As the distance from the electrode increases, alginate has a greater tendency to remain in solution due to the reduced presence of calcium ions. (B) An L-shaped patterned ITO electrode was used to electrodeposit alginate. A PDMS well was fixed to the electrode to contain a green fluorescently-labeled alginate (1%) and CaCO3 (0.5%) deposition solution. After electrodepositing for 2 min. at a current density of 3A/m2, the electroaddressed alginate hydrogel was imaged by fluorescence microscopy.
Figure 2. Codeposition of cell populations. (A) Scheme showing interaction between two E. coli strains: One population produces autoinducer-2 (AI-2), a signaling molecule, and is termed “AI-2 sender.” The other population, termed “AI-2 receiver,” is a reporter of AI-2; upon receipt of AI-2 by diffusion from the sender, it expresses the red fluorescent protein dsRed. (b) Red fluorescence image of electrode pair with the AI-2 sender population codeposited with alginate on the left electrode and AI-2 receiver population codeposited with alginate on right electrode. Magnified view demonstrates the dsRed expression of only the AI-2 receivers.
Figure 3. Chitosan electrodeposition. (A) Scheme showing the pH-dependent electrodeposition of chitosan. Water electrolysis at a cathodically biased electrode causes a localized high pH (shown by a localized color change of a pH indicator dye near the cathode in micrograph) which stimulates the sol-gel transition of chitosan in this region. (B) The amines present on chitosan give it pH-responsive properties. Above a pH of 6.3 (pKa of chitosan) the amines are deprotonated, facilitating a transition from its protonated soluble form to its insoluble gel form. (C) A patterned gold electrode was used to electrodeposit chitosan. The electrode, connected cathodically to the power supply, was immersed into a green fluorescently-labeled chitosan (0.8%) deposition solution. After electrodepositing for 2 min. at a current density of 4 A/m2, the electroaddressed chitosan film was imaged by fluorescence microscopy.
Figure 4. Electrochemical transduction with a functionalized chitosan film. (A) Schematic showing the set-up of a three electrode system. Functionalized chitosan film serves as the working electrode, a platinum wire as the counter electrode and Ag/AgCl as the reference electrode. Electrochemical transduction of glucose proceeds through the enzymatic and electrochemical reactions shown where produced hydrogen peroxide can be oxidized and detected at the working electrode. (B) Cyclic voltammagram (CV) at electrode with a chitosan film containing electrochemically conjugated glucose oxidase (GOx) shows a strong anodic signal in a 5 mM glucose solution. A film containing no GOx served as a control and displayed no signal in the same solution. (C) A standard curve between anodic current and the glucose concentration displays a near linear relationship (each aliquot increased the glucose concentration by 4 mM and also increased the current amplitude in the inset graph in a step-wise manner).
Figure 5. Protein functionalization using enzymatic assembly. (A, upper) Scheme showing tyrosine-tagged “AI-2 Synthase” being covalently tethered to a chitosan film by tyrosinase assembly. The tyrosine residues become oxidized to O-quinones by tyrosinase action and may react with amine groups on the chitosan film, forming a covalent bond. (A, lower) The AI-2 Synthase generates AI-2 from a substrate (SAH); the receiver cells report the generated AI-2 by dsRed fluorescence expression. (B) Fluorescence images showing a chitosan film on gold, functionalized with blue-labeled AI-2 Synthase. Adjacently, AI-2 receiver cells are codeposited with alginate on ITO. After addition of the enzymatic substrate to the well and incubation, the AI-2 receiver cells express dsRed.
Name of the component | Company | Catalogue number |
Power Supply | Keithley | SourceMeter 2400 |
Three electrode potentiostat | CH Instruments | Potentiostat/Galvanostat 600D |
RE-5B Ag/AgCl Reference Electrode with Flexible Connector | BASi | MF-2052 |
Gold coated silicon wafer, 500um Si, 12nM Cr, 120nM Au, SiO2 for insulation | custom fabricated | |
Indium Tin oxide coated glass slide, rectangular, 8-12 ohm resist | Sigma-Aldrich | 578274 |
Platinum sheet/foil (0.002 in) | Surepure Chemetals | 1897 |
Slim Line 2″ Alligator Clips | RadioShack | 270-346 |
Multi-Stacking Banana Plug Patch Cord | TSElectronic | B-36-02 B-24-02 |
SYLGARD 184 silicone elastomer kit | Dow Corning | NC9020938 From Fischer |
Fluorescecence stereomicroscope | Olympus | MVX10 MacroView |
cellSens Standard | Olympus | version 1.3 |
Table 1. Electrodeposition and fluorescence visualization equipment.
Name of the reagent | Company | Catalogue number |
Chitosan, medium molecular weight | Sigma-Aldrich | 448877 |
Hydrochloric Acid, ARISTAR. ACS, NF, FCC Grade | VWR | BDH3030 |
Sodium Hydroxide, Solution. 10.00N | VWR | VW3247 |
Alginic acid, sodium salt | Sigma-Aldrich | 180947 |
Multifex-MM Precipitated Calcium Carbonate, 70nm particles |
Speciality Minerals Inc. |
100-3630-3 |
Table 2. Chitosan and alginate solution reagents.
Name of the reagent | Company | Catalogue number |
Calcium chloride, dihydrate | J.T. Baker | 0504 |
Sodium Chloride, Certified ACS crystalline |
Fischer Scientific |
S271 |
Potassium Phosphate Monobasic, anhydrous | Sigma-Aldrich | P9791 |
Potassium Phosphate Dibasic, anhydrous | Sigma- Aldrich | P3786 |
Phosphate Buffered Saline | Sigma- Aldrich |
P4417 |
Table 3. Other solution components and buffer reagents.
Name of the reagent | Company/Source | Catalogue number |
Glucose oxidase from aspergillus niger | Sigma-Aldrich | G2133 |
Tyrosinase from mushroom | Sigma-Aldrich | T3824 |
LB broth, Miller (granulated) | Fischer Scientific | BP9723-2 |
“AI2-Synthase” (HGLPT) | Lab stock 16 | |
W3110 wildtype cells | Lab stock 30 | |
MDAI2 + pCT6-lsrR–ampr + pET-dsRed–kanr cells | Lab stock 30 | |
FluoroSpheres: 1μm diameter, Ex/Em: 505/515 | Invitrogen | F8765 |
5-(and-6)-carboxyrhodamine 6G succinimidyl ester, Ex/Em: 525/560 | Invitrogen | C-6157 |
DyLight antibody labeling kit, 405 | Thermo Scientific | PI-53020 |
Table 4. Enzymes, cells, and other functionalization reagents.
Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission.
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.
Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission.
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.
Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission.
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.